Bottom Line:
In the aqueous humor of the noninjected eye, maximum concentration of bevacizumab was achieved at day 8 (1.6125 ng/mL) and declined (to 0.11 ng/mL) at 4 weeks.The vitreous half-life of 1.25 mg/0.05 mL intravitreal bevacizumab was 6.61 days in this rabbit model.Very low concentrations of bevacizumab were measured in the fellow noninjected eye.

Affiliation: Laboratory for Experimental Surgery and Surgical Research 'N.S.Christeas', School of Medicine, National and Kapodistrian University of Athens, 15b AgiouThoma Street, 11527, Athens, Greece. csinapis@yahoo.gr

ABSTRACT

Purpose: To describe the pharmacokinetics of intravitreal bevacizumab (Avastin®) in rabbits.

Methods: The right eye of 20 rabbits was injected intravitreally with 1.25 mg/0.05 mL bevacizumab. Both eyes of four rabbits each time were enucleated at days 1, 3, 8, 15, and 29. Bevacizumab concentrations were measured in serum, aqueous humor, and vitreous.

Results: Maximum vitreous (406.25 μg/mL) and aqueous humor (5.83 μg/mL) concentrations of bevacizumab in the right eye were measured at day 1. Serum bevacizumab concentration peaked at day 8 (0.413 μg/mL) and declined to 0.032 μg/mL at 4 weeks. Half-life values in right vitreous, right aqueous humor, and serum were 6.61, 6.51, and 5.87 days, respectively. Concentration of bevacizumab in the vitreous of the noninjected eye peaked at day 8 (0.335 ng/mL) and declined to 0.218 ng/mL at 4 weeks. In the aqueous humor of the noninjected eye, maximum concentration of bevacizumab was achieved at day 8 (1.6125 ng/mL) and declined (to 0.11 ng/mL) at 4 weeks.

Conclusion: The vitreous half-life of 1.25 mg/0.05 mL intravitreal bevacizumab was 6.61 days in this rabbit model. Maximum concentrations of bevacizumab were reached at day 1 in both vitreous and aqueous humor of the right eye and at day 8 in the serum. Very low concentrations of bevacizumab were measured in the fellow noninjected eye.

Mentions:
Bevacizumab concentrations were measured using enzyme-linked immunosorbent assay (ELISA). Two ELISA methods were used, one of low sensitivity (LS ELISA, linear range: 5 ng/mL to 0.1 μg/mL, Figure 1A) and one of high sensitivity (HS ELISA, linear range: 10pg/mL to 5 ng/mL, Figure 1B). ELISA plates (Costar high binding) were coated with 100 μL/well of rec-hVEGF (R&D Systems, Minneapolis, MN) at a concentration of 0.2 μg/mL in carbonate-bicarbonate buffer (pH = 9.6). After washing with PBS (200 μL/well), the plates were blocked with 200 μL/well of bovine albumin 2% in PBS (BB: blocking buffer). Afterwards, the plates were washed and the samples were added (100 μL/well) in various dilutions ranging from 1:10,000 (vitreous/right eye day1) to 1:1 (vitreous/left eye day 29) and incubated for 2 hours at room temperature (RT). The plates were then washed again and for i) LS ELISA: 100 μL/well of anti-human Fab specific antibody conjugated to horse radish peroxidase (HRP) (Sigma-Aldrich, St Louis, MO) (1:1200 in BB) was added to the wells. After washing, 100 μL of ABTS [2, 2′-azino-bis (3-ethylbenzthiazoline-6-sulfonicacid)] substrate was added to the wells and the color development was measured at 405 nm, ii) HS ELISA: 100 μL/well of anti-human Fab specific antibody conjugated to alkaline phosphatase (AP) (Jackson Immuno research, West Grove, PA) (1:1200 in BB) was added to the wells. Subsequently, the plates were washed four times with PBS and the Invitrogen’s ELISA amplification system was used to enhance the detection of bevacizumab. Briefly, the plates were incubated with 50 μL/well of substrate (reduced NADPH) for 20 minutes followed by the addition of 50 μL/well of amplifier (alcohol dehydrogenase and diaphorase). The color was quantified at 490 nm.

Mentions:
Bevacizumab concentrations were measured using enzyme-linked immunosorbent assay (ELISA). Two ELISA methods were used, one of low sensitivity (LS ELISA, linear range: 5 ng/mL to 0.1 μg/mL, Figure 1A) and one of high sensitivity (HS ELISA, linear range: 10pg/mL to 5 ng/mL, Figure 1B). ELISA plates (Costar high binding) were coated with 100 μL/well of rec-hVEGF (R&D Systems, Minneapolis, MN) at a concentration of 0.2 μg/mL in carbonate-bicarbonate buffer (pH = 9.6). After washing with PBS (200 μL/well), the plates were blocked with 200 μL/well of bovine albumin 2% in PBS (BB: blocking buffer). Afterwards, the plates were washed and the samples were added (100 μL/well) in various dilutions ranging from 1:10,000 (vitreous/right eye day1) to 1:1 (vitreous/left eye day 29) and incubated for 2 hours at room temperature (RT). The plates were then washed again and for i) LS ELISA: 100 μL/well of anti-human Fab specific antibody conjugated to horse radish peroxidase (HRP) (Sigma-Aldrich, St Louis, MO) (1:1200 in BB) was added to the wells. After washing, 100 μL of ABTS [2, 2′-azino-bis (3-ethylbenzthiazoline-6-sulfonicacid)] substrate was added to the wells and the color development was measured at 405 nm, ii) HS ELISA: 100 μL/well of anti-human Fab specific antibody conjugated to alkaline phosphatase (AP) (Jackson Immuno research, West Grove, PA) (1:1200 in BB) was added to the wells. Subsequently, the plates were washed four times with PBS and the Invitrogen’s ELISA amplification system was used to enhance the detection of bevacizumab. Briefly, the plates were incubated with 50 μL/well of substrate (reduced NADPH) for 20 minutes followed by the addition of 50 μL/well of amplifier (alcohol dehydrogenase and diaphorase). The color was quantified at 490 nm.

Bottom Line:
In the aqueous humor of the noninjected eye, maximum concentration of bevacizumab was achieved at day 8 (1.6125 ng/mL) and declined (to 0.11 ng/mL) at 4 weeks.The vitreous half-life of 1.25 mg/0.05 mL intravitreal bevacizumab was 6.61 days in this rabbit model.Very low concentrations of bevacizumab were measured in the fellow noninjected eye.

Affiliation:
Laboratory for Experimental Surgery and Surgical Research 'N.S.Christeas', School of Medicine, National and Kapodistrian University of Athens, 15b AgiouThoma Street, 11527, Athens, Greece. csinapis@yahoo.gr

ABSTRACT

Purpose: To describe the pharmacokinetics of intravitreal bevacizumab (Avastin®) in rabbits.

Methods: The right eye of 20 rabbits was injected intravitreally with 1.25 mg/0.05 mL bevacizumab. Both eyes of four rabbits each time were enucleated at days 1, 3, 8, 15, and 29. Bevacizumab concentrations were measured in serum, aqueous humor, and vitreous.

Results: Maximum vitreous (406.25 μg/mL) and aqueous humor (5.83 μg/mL) concentrations of bevacizumab in the right eye were measured at day 1. Serum bevacizumab concentration peaked at day 8 (0.413 μg/mL) and declined to 0.032 μg/mL at 4 weeks. Half-life values in right vitreous, right aqueous humor, and serum were 6.61, 6.51, and 5.87 days, respectively. Concentration of bevacizumab in the vitreous of the noninjected eye peaked at day 8 (0.335 ng/mL) and declined to 0.218 ng/mL at 4 weeks. In the aqueous humor of the noninjected eye, maximum concentration of bevacizumab was achieved at day 8 (1.6125 ng/mL) and declined (to 0.11 ng/mL) at 4 weeks.

Conclusion: The vitreous half-life of 1.25 mg/0.05 mL intravitreal bevacizumab was 6.61 days in this rabbit model. Maximum concentrations of bevacizumab were reached at day 1 in both vitreous and aqueous humor of the right eye and at day 8 in the serum. Very low concentrations of bevacizumab were measured in the fellow noninjected eye.